Part:BBa_K3332089

pLtetO-1-RBS1-LacI-ssrAtag(mf-lon)-terminator-pTrc-2 derivative-RBS-ECFP-terminator

With this part, the LacI protein can be tested by observing the fluorescence intensity.

Usage and Biology

This part can be used to test that if the LacI protein can repress the pTrc-2 derivative promoter. With the expression of LacI, the bacteria have little fluorescence.

Fig 1. pLtetO-1_RBS1_LacI_terminator_pTrc-2-derivative_RBS(B0034)_ecfp_terminator(BBa_K3332089)

Characterization

We use pTrc-2 derivative_E0420_pUC57(BBa_K3332087),pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57 and pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57(BBa_K3332089) to characterize pTrc-2 promoter and pTrc-2 derivative promoter. The agarose gel electrophoresis images are below:

Fig 2. pTrc-2 derivative_E0420_pUC57(BBa_K3332087) digested by EcoR I and Pst I (about 1018 bp).

Fig 3. pLtetO-1_RBS1_lacI_B0015_pTrc-2_E0420_pUC57(BBa_K3332088） digested by Pst I (about 5086 bp).

Fig 4. pLtetO-1_RBS1_lacI_B0015_pTrc-2 derivative_E0420_pUC57（BBa_K3332089） digested by Pst I (about 5125 bp).

note: E0420 is equal to B0034_E0020_B0015.

Protocol:

1.Preparation of stock solution:dissolve IPTG in absolute alcohol to make 1000× stock solution.

2.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.

3.Add 4 mL of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 100 μL IPTG stock solution into the induction group when OD₆₀₀ increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 1 minute. Remove supernatant and add 500 µL sterile PBS to resuspend.

7.Measure the fluorescence intensity(ECFP)and corresponding OD₆₀₀ by 96-well plate reader, then calculate the fluorescence / OD value of each group. Here is the result:

Fig 5. Fluorescence intensity/OD for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form．

The strength of pTrc2-derivative and pTrc2 are contrasted. In the figure, pTrc2-derivative are used as the negative control group, pTrc2-derivative_E0420(ECFP)(BBa_K3332087) are used as the positive control group while pLtetO-1_LacI_pTrc2_E0420(ECFP) and pLtetO-1_LacI_pTrc2-derivative_E0420(ECFP) are both experimental groups.

We can see, after adding IPTG to induce the two promoters, the fluorescence intensity are both improved. The change of fluorescence intensity after induction of pLtetO-1-LacI-pTrc2-E0420(ECFP) group is larger than pLtetO-1-LacI-pTrc2-derivative-E0420(ECFP) group, so we can confirm that LacI has a weak inhibitory effect on pTrc-2 promoter and a strong inhibitory effect on pTrc-2 derivative promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1489
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979